The firm said the Heathrow Express was running "normally"
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Rail bosses have been criticised for using managers to drive trains during a strike over pay and conditions.
The Rail Maritime and Transport union (RMT) said lives were being put at risk by using "inadequately-trained managers" on the Heathrow Express.
The company denied the union's accusations, saying only experienced staff were driving trains.
It added a normal service was running on the trains which run from Heathrow Airport to Paddington station.
Up to 80 workers voted 12-1 for a 24-hour walkout on Thursday and Monday.
Heathrow Express said only a few of the 275 workforce had failed to report for duties.
But RMT general secretary Bob Crow, said: "Our members are out solidly today and if the company keeps their head buried in the sand they will be out solidly on Monday.
"Using inadequately-trained managers to run the trains will not solve this dispute but it will put lives at risk."
A company spokesman said: "Heathrow Express rejects the RMT's claims that we putting peoples lives at risk by using inadequately trained managers to run trains.
"Only experienced staff who have the correct level of competence are allowed to undertake safety critical tasks such as driving trains.
"We are pleased to say that the RMT's strike action has not had any impact on our services."